Scientific description: A comprehensive description of the identities, concentrations, and interactions of proteins found in hair bundles, the mechanically sensitive organelles of the inner ear, will allow a thorough molecular description of mechanotransduction and hair-bundle assembly. Mass spectrometry methods are now sufficiently sensitive to apply to scarce hair bundles; indeed, our preliminary experiments have identified 56 proteins from a preparation of purified chicken bundles. In this project, we will (1) identify those proteins that are enriched in hair bundles using iTRAQ differential labeling and liquid-chromatography-tandem mass spectrometry (LC-MS/MS), (2) determine the concentrations of key bundle proteins using multiple reaction monitoring techniques and LC-MS/MS, and (3) measure the concentrations of critical bundle proteins during development using both sets of methods. These three aims will serve as pilot experiments to allow systematic application of tandem mass spectrometry to biochemical characterization of hair-bundle function. This project will allow development of sufficiently sensitive mass spectrometry methods to achieve our eventual goal of routinely measuring the concentrations and modification states of hundreds of hair bundle proteins in a single LC-MS/MS experiment. Lay description: Sound excites hair cells, which convert sound energy into electrical signals in the brain by a process called mechanotransduction. Hair cells are remarkably sensitive, but that sensitivity translates into fragility; moreover, once hair cells die, they are not replaced. To understand how hair cells die and how we might grow new ones, we need to understand what their mechanically sensitive structure - the hair bundle - is made of and how it is put together. This project is an essential first step in understanding what makes up a hair bundle. [unreadable] [unreadable] [unreadable]